Kallikrein is an enzyme which functions physiologically in the formation of the hypotensive peptides bradykinin and kallidin by hydrolytic cleavage of the precursor peptide kininogen. The enzyme hydrolyzes peptide bonds on the carboxyl side of arginine or lysine residues and thus resembles trypsin and other proteases having a serine at the active site, such as thrombin, urokinase, and plasmin. There are two main groups of kallikreins, plasma and glandular. Urinary kallikrein has similar characteristics to a glandular kallikrein. The urinary enzyme is of special interest because it is implicated in blood pressure regulation and regulation of sodium balance. The assay of human urinary kallikrein is of clinical significance in the diagnosis of hypertension, in determining an appropriate course of treatment and in monitoring the effects of medication.
Serum and urinary kallikreins are immunologically distinguishable and have different pH optima and substrate specificities. Human urinary kallikrein is immunologically distinct from rat or dog urinary kallikrein. The enzyme is found only in minute amounts in human urine. Development of an assay for human urinary kallikrein activity requires the specific development of a suitable substrate capable of providing sufficient specificity and sensitivity. Compounds such as benzoylarginine ethyl ester and tosylarginine methyl ester, although hydrolyzed by the enzyme, are unsuitable because of the many known enzymes of trypsin-like specificity which also hydrolyze the substrates. In general, substrates whose hydrolysis is measured spectrophotometrically are unsuitable since such measurements do not provide sufficient sensitivity for convenient measurement of the minute amounts of human urinary kallikrein encountered in clinical practice.
A variety of substrates suitable for kallikrein have been discovered among peptide analogs of the C-terminal portion of bradykinin. Svendsen, U.S. Pat. No. 4,016,042 disclosed benzoyl-ProPheArg-p-nitroanilide as a substrate for spectrophotometric assay of serum kallikrein. (All amino acid residues are in the L-configuration herein, unless otherwise specified.) The following abbreviations are employed:
______________________________________ Tos Tosyl Pro Proline Phe Phenylalanine Arg Arginine Ser Serine Boc t-butoxycarbonyl Aoc amyloxycarbonyl ______________________________________
Hydrolysis of the nitroanilide moiety results in a spectrophotometric change detectable where micromolar amounts of product are accumulated. However, the substrate is inactive with human urinary kallikrein, at least where present in amounts currently needed for assay.
It was suggested by Day, A. R., Chung, A. and Ryan, J. W., Agents and Actions 6, 421 (1976), that the compounds Boc-ProPheArg-orthohydroxyanilide and Boc-ProPheArg-p-iodoanilide could be converted into suitable radioassay substrates by [.sup.125 I] iodonation of the former compound and catalytic tritiation by dehalogenation of the latter. For reasons not presently understood, however, the catalytic tritiation of the p-iodoanilide has proven to be a difficult reaction, with low yields and consequent low specific activity, under the best of circumstances.
Attention has recently focused on kallikrein substrate peptides having at least one D-amino acid in the sequence. For example, German O.S. No. 2629067 discloses derivatives of D-ProPheArg, for the assay of serum kallikrein. Substrates of this sort have been found to provide a more sensitive assay than the L-enantiomers, for the measurement of serum kallikrein. The success of the D-amino acid containing peptides has prompted further research to discover even more effective substrates.